This proposal will focus on studies of voltage-gated ion channels, specifically: 1) charge movement in a voltage sensing domain, with an emphasis on the largely unexplored intracellular aqueous vestibules near the S4 segment, and 2) open-channel block and permeation, especially in voltage-gated sodium channels, where knowledge of these processes is at a relatively superficial level. The first aim is to examine the mechanisms of charge movement in domain IV (D4) of the sodium channel and the roles played by transmembrane segments in the vicinity of its S4 segment, and by dynamic aqueous vestibules. To this end we will use a combination of mutagenesis, cysteine accessibility scanning, cysteine crosslinking, and theoretical analysis of electrostatic potentials in the crevices near S4 segments. Ultimately we will create a dynamic model of topological and electrostatic profiles of aqueous crevices in the voltage sensing domain of D4. The second aim is to test proposed but unanswered hypotheses about the roles of aromatic residues in permeation and block, and of a pore-loop lysine essential for selectivity of sodium channels. To do this, we will insert unnatural amino acids into potassium and sodium channels by the 'in vivo suppression methodology'. We will test the role of cation-pi interactions between extracellular blockers and aromatic residues at both extracellular and intracellular locations in the permeation pathway. Cationic blockers will include extracellular toxins (tetrodotoxin, saxitoxin, and mu-conotoxin) and intracellular local anesthetics. We will test for a role of the pi electrons of critical aromatic residues in selectivity, conductance, block, and inactivation. We will also explore the effects of subtle structural alterations of a critical lysine residue in the pore loop of D3 in sodium channels. After nearly two decades of structure-function studies on voltage-gated ion channels, proteins that underlie excitability in nerve and muscle cells, a number of fundamental issues remain unresolved, including how these channels respond robustly to small changes of membrane potential and how sodium channels select their namesake ion over others. There are also gaping holes in our understanding of the interactions between these ion channels and a number of toxins and pharmacological agents. The proposed studies should help in the design of therapeutic drugs to treat a variety of malfunctions in excitability.